Optimized DNA and protein sequence of an antibody to improve quality and yield of bacterially expressed antibody fusion proteins

ABSTRACT

Object matter of the invention is an optimized DNA sequence encoding the scFv(FRP5) antibody fragment. This novel sequence prevents the generation of the undesired by-product in the context of an scFv(FRP5)-ETA fusion protein, and possibly also other bacterially expressed scFv(FRP5)-containing fusion proteins. The DNA sequence of the scFv(FRP5) domain of scFv(FRP5)-ETA was modified by exchanging a distinct codon, thereby preventing an otherwise possible internal start of protein translation.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/949,580 filed Jul. 13, 2007, the contents of which areincorporated by reference herein in their entirety.

The antibody-toxin scFv(FRP5)-ETA is a recombinant fusion proteincomposed of a single-chain antibody fragment derived from theErbB2-specific antibody FRP5, via gene fusion linked to a truncatedfragment of Pseudomonas exotoxin A. High and selective antitumoralactivity of scFv(FRP5)-ETA against ErbB2 expressing cancer cells invitro, in animal models and in cancer patients has been described indetail in the literature. Production of scFv(FRP5)-ETA by bacterialexpression in E. coli using current methodology results, in addition tothe major product of intact scFv(FRP5)-ETA, also in a truncatedscFv(FRP5)-ETA fragment as a by-product. Complete elimination of thisundesired fragment using classical protein purification techniques hasso far not been achieved.

Object matter of the invention is an optimized DNA sequence encoding thescFv(FRP5) antibody fragment. This novel sequence prevents thegeneration of the undesired by-product in the context of anscFv(FRP5)-ETA fusion protein, and possibly also other bacteriallyexpressed scFv(FRP5)-containing fusion proteins. The DNA sequence of thescFv(FRP5) domain of scFv(FRP5)-ETA was modified by exchanging adistinct codon, thereby preventing an otherwise possible internal startof protein translation.

BACKGROUND OF THE INVENTION

Epithelial cells of most organs typically express the ErbB2 (HER2)receptor tyrosine kinase at low levels. However, in several types ofcarcinomas, ErbB2 expression is strongly enhanced, often as a result ofgene amplification. Due to this preferential expression in many tumorsof epithelial origin, its accessibility from the extracellular space,and its involvement in the transformation process, the ErbB2 receptortyrosine kinase is a preferred target for directed cancer therapy.

Based on a truncated Pseudomonas exotoxin A derivative lacking thetoxin's endogenous cell binding domain, a recombinant toxin wasdeveloped that employs a single-chain Fv antibody fragment of theErbB2-specific monoclonal antibody FRP5 for targeting of the toxin toErbB2 (1). In in vitro cell killing experiments, this bacteriallyexpressed scFv(FRP5)-ETA molecule displayed potent antitumoral activityagainst a wide range of established and primary human tumor cells,including breast and ovarian carcinomas (1-3), squamous cell carcinomas(4, 5) and prostate carcinomas (6). In experimental animalsscFv(FRP5)-ETA effectively inhibited growth of established human tumorxenografts (1, 3-5) and murine and rat tumor cells stably transfectedwith human c-erbB2 constructs (7, 8). In cancer patients, intratumoralinjection of scFv(FRP5)-ETA into cutaneous lesions of ErbB2 expressingtumors resulted in a response rate of 60%, with complete regression ofinjected tumor nodules observed in 40%, and partial reduction in thesize of injected tumors in another 20% of patients (9). In a recentphase I clinical study, maximum tolerated dose (MTD), dose limitingtoxicity and pharmacokinetic parameters of intravenously injectedscFv(FRP5)-ETA were determined (10). Thereby three out of 18 patientsshowed stable disease, and in another three patients clinical signs ofactivity in terms of signs and symptoms were observed.

SUMMARY OF THE INVENTION

Preparations of therapeutic proteins for the treatment of human patientsmust meet very high standards of purity and homogeneity to qualify forapproval by regulatory authorities. By-products contaminatingpreparations of the active compound may cause adverse events in apatient, such as toxic reactions, and/or the induction of undesiredimmune responses. Therefore, such by-products must be removed to theextent technically possible, and for any remaining by-products, possiblebiological activities or the absence thereof must be individuallydemonstrated. As a consequence, production costs will dramaticallyincrease due to the requirement for sophisticated and expensivepurification techniques used to remove such undesired by-products,and/or due to additional testing that is required if a particularby-product cannot be removed.

For production of the scFv(FRP5)-ETA antibody-toxin for in vivoapplications, so far mainly the bacterial expression vector pSW220-5 wasused (7). This plasmid encodes a fusion protein composed of theErbB2-specific scFv antibody fragment scFv(FRP5) derived from monoclonalantibody FRP5 (11, 12), genetically fused to truncated Pseudomonasexotoxin A (ETA), representing amino acid residues 252-613 of thewildtype toxin. In addition, the scFv(FRP5)-ETA expression unit inplasmid pSW220-5 includes sequences for two hexahistidine clusters(His₆) and an N-terminal FLAG tag for purification and detection of theprotein (FIG. 1A). Protein preparations from bacterial expressioncultures transformed with pSW220-5 contain a major product correspondingto full-length scFv(FRP5)-ETA, and a major by-product (about 10%) thatmigrates directly below the main band. Both protein bands can bedetected in purified protein preparations by Coomassie-staining ofSDS-PAA gels (see FIG. 2A, left lane), and with ETA-specific antibodiesin immunoblot experiments (data not shown). Also previous immunoblotexperiments revealed that the by-product is being recognized byantibodies to the exotoxin A portion of scFv(FRP5)-ETA. This by-producthas therefore been thought to be generated during or after expression byprotein degradation of full-length scFv(FRP5)-ETA by bacterialproteases. Using standard protein purification techniques, it has notbeen possible so far to remove this truncated protein fragment.

DETAILED DESCRIPTION OF THE INVENTION AND OF EXEMPLARY EMBODIMENTS

It is an object of the invention to prevent the generation of thistruncated fragment of scFv(FRP5)-ETA without affecting thetherapeutically relevant biological activities of the full-lengthprotein. Upon application of this invention, this undesired by-productcan no longer form during bacterial expression. Therefore proteinpreparations of higher purity can now easily be produced.

This invention covers the modification of the expression unit encodingscFv(FRP5)-ETA in such a way, that a homogeneous protein preparation canbe obtained from bacterial expression cultures, which lacks thetruncated by-product mentioned above. In contrast to previousconsiderations, we hypothesized that the by-product may not be generatedby proteolytic degradation of full-length protein, but may be the resultof an alternative start of protein translation from an internal AUGcodon within the scFv(FRP5) sequence of the scFv(FRP5)-ETA mRNA.

In a first aspect, the invention relates to a polypeptide comprising afirst amino acid sequence which comprises amino acids 2-120 of SEQ IDNO:11, and a second amino acid sequence which comprises amino acids136-242 of SEQ ID NO:11, wherein said first and second amino acidsequence are linked by a peptide spacer group. Preferably, thepolypeptide of the invention comprises the following structure:V_(H)-Sp-V_(L),whereinV_(H) is the first amino acid sequence,Sp is the peptide spacer group, andV_(L) is the second amino acid sequence.

Accordingly, the first amino acid sequence is usually at the N-terminalend of the polypeptide. The polypeptide of the invention is usually asingle-chain antibody wherein the heavy chain variable domain and thelight chain variable domain are linked by way of a spacer group,preferably a peptide. The first amino acid sequence in the polypeptideof the invention represents the heavy chain variable domain, and thesecond amino acid sequence represents the light chain variable domain.Most preferred is a single-chain antibody wherein the heavy chainvariable domain is located at the N-terminus of the recombinantantibody.

The first amino acid sequence comprises amino acids 2-120 of SEQ IDNO:11, preferably comprises amino acids 1-120 of SEQ ID NO:11, and morepreferably consists of amino acids 1-120 of SEQ ID NO:11. In a morepreferred embodiment, the first amino acid sequence comprises aminoacids 2-120 of SEQ ID NO: 1, preferably comprises amino acids 1-120 ofSEQ ID NO:1, and more preferably consists of amino acids 1-120 of SEQ IDNO:1.

The second amino acid sequence comprises amino acids 136-242 of SEQ IDNO:11, but preferably consists of amino acids 136-242 of SEQ ID NO:11.

The peptide spacer group may have a length of from 3 to 30 amino acids,preferably of from 5 to 25 amino acids, more preferably of from 10 to 20amino acids, most preferably of about 15 amino acids (e.g. 13, 14, 15,16 or 17 amino acids). It is also preferred that the peptide spacergroup consists of amino acids selected from glycine and serine.Particularly preferred is an embodiment, wherein the spacer group is the15 amino acid peptide consisting of three repetitive subunits ofGly-Gly-Gly-Gly-Ser.

The polypeptide of the invention preferably comprises the amino acidsequence as shown in SEQ ID NO:11, but more preferably comprises theamino acid sequence as shown in SEQ ID NO:1.

The amino acid Xaa in SEQ ID NO:11 may be any amino acid exceptmethionine or be absent. In the latter case, the amino acids atpositions 91 and 93 are linked directly to each other via a peptidebond. Xaa in SEQ ID NO:11 may be any amino acid except methionine,including naturally occurring amino acids, non-naturally occurring aminoacids and modified amino acids. When Xaa is a naturally occurring aminoacid, Xaa may be alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,asparagine, proline, glutamine, arginine, serine, threonine, valine,tryptophane, tyrosine or selenocysteine. Preferably, Xaa is selectedfrom the group consisting of serine, alanine, threonine and cysteine.Most preferably, Xaa is serine. When Xaa is a non-naturally occurring ormodified amino acid, possible meanings of Xaa include, but are notlimited to ornithine, norleucine, norvaline, hydroxyproline,hydroxylysine, ethylglycine and ethylasparagine. Xaa may also be anymodified amino acid as defined in table 4 of WIPO Standard ST.25, whichis incorporated herein by reference.

Alternatively, Xaa may be absent which means that the methionine atposition 92 of the FRP5 sequence (see e.g. SEQ ID NO:9) has been deletedand is not replaced by another amino acid. According to that embodiment,the polypeptide of the invention may comprise the amino acid sequence asshown in SEQ ID NO:12.

The single-chain recombinant antibody may further comprise an effectormolecule and/or signal sequences facilitating the processing of theantibody by the host cell in which it is prepared.

Effector molecules considered are those useful for therapeutic ordiagnostic purposes, for example enzymes causing a detectable reaction,e.g. phosphatase, such as alkaline phosphatase from E. coli or mammalianalkaline phosphatase, e.g. bovine alkaline phosphatase, horseradishperoxidase, beta-D-galactosidase, glucose oxidase, glucoamylase,carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenaseor glucose-6-phosphate, a peptide having particular binding properties,e.g. streptavidin from Streptomyces avidinii strongly binding to biotin,or enzymes, toxins or other drugs attacking the cells to which theantibody is bound, e.g. a protease, a cytolysin or an exotoxin, forexample ricin A, diphtheria toxin A, or Pseudomonas exotoxin. In thefollowing a single-chain recombinant antibody further comprising aneffector molecule is referred to as fusion protein or intended to bewithin the meaning of the terms “single chain (recombinant) antibody” or“recombinant antibody”, if appropriate. The effector molecule may be apolypeptide having cell killing activity. Cell killing activity may bedetermined according to Example 3 of this application.

The term effector molecule also includes biologically active variants ofthe above-mentioned proteins, e.g. variants produced from a DNA whichhas been subjected to in vitro mutagenesis, with the provision that theprotein encoded by said DNA retains the biological activity of thenative protein. Such modifications include an addition, exchange ordeletion of amino acids, the latter resulting in shortened variants. Forexample, an enzyme, such as phosphatase, may be prepared from a DNAwhich has been modified to facilitate the cloning of the encoding gene,or an exotoxin, such as Pseudomonas exotoxin, may be prepared from a DNAwhich has been mutated to delete the cell binding domain and/or toenhance or reduce its cell killing potential.

The effector polypeptide may comprise the amino acids 245-606 of SEQ IDNO:1.

The term effector molecule also includes chemical entities having cellkilling activity. Cell killing activity may be determined according toExample 3 of this application. Such chemical entities include, but arenot limited to, chemotherapeutic drugs, cytotoxic compounds andcytostatic compounds. Examples of chemotherapeutic drugs and cytotoxiccompounds are

-   -   alkylating agents    -   cytotoxic antibiotics    -   antimetabolites    -   vinca alkaloids and etoposide    -   others

Alkylating agents react with nucleophilic residues, such as the chemicalentities on the nucleotide precursors for DNA production. They affectthe process of cell division by alkylating these nucleotides andpreventing their assembly into DNA. Suitable alkylating agents includeMustargen, Estramustinphosphate, Melphalan, Chlorambucil, Prednimustin,Cyclophosphamide, Ifosfamid, Trofosfamid, Busulfan, Treosulfan,Thiotepa, Carmustin (BCNU), Lomustin (CCNU), Nimustin (ACNU),Dacarbazine (DTIC), Procarbazine, Cisplatin, and Carboplatin.

Cytotoxic antibiotics act by directly inhibiting DNA or RNA synthesisand are effective throughout the cell cycle. Suitable cytotoxicantibiotics include Actinomycin D, Daunorubicin, Doxorubicin,Epirubicin, Idarubicin, Mitoxantron, Bleomycin, Mitomycin C, Irinotecan(CPT-11), and Topotecan.

Antimetabolites interfere with cellular enzymes or natural metabolitesthat are involved in the process of cell division, thus disrupting thedivision of the cell. Suitable Antimetabolites include Methotrexate,6-Mercaptopurine, 6-Thioguanine, Pentostatin, Fludarabinphosphate,Cladribine, 5-Fluorouracil, Capecitabine, Cytarabin, Gemcitabine, andHydroxyurea.

Plant alkaloids and etoposides are agents derived from plants. Theyinhibit cell replication by preventing the assembly of the cell'scomponents that are essential to cell division (e.g. Vinca alkaloids;Etoposide). Suitable alkaloids and etoposides include Vincristin,Vinblastin, Vindesin, Etoposide (VP16), Teniposide (VM26).

The group of compounds labeled ‘Others’ is made up primarily of taxanes(e.g. Paclitaxel, Taxol, Docetaxel, Taxotere) and metal complexes (e.g.cis Platinum), and signal transduction inhibitors (STIs), inhibitors ofspecific enzyme functions, including but not limited to histonedeacetylase inhibitors, kinase and protease inhibitors. The histonedeacetylase (HDAC) inhibitor may be selected from the group consistingof vorinostat, belinostat, PCI-24781, CHR-3242, JNJ-16241199, MGCD-0103,romidepsin, MS-275, butyrate, valproic acid and combinations thereof.The kinase inhibitor may be selected from the group consisting ofimatinib, cedarinib, gefitinib, vandetanib, sarafenib, danatinib,lestaurtinib, enzastaurin, pazopanib, alvocidib, nilotinib, vatalinib,erlotinib, suninib and combinations thereof. The protease inhibitor maybe selected from the group consisting of WX-UK1, bortezomib andcombinations thereof.

Alternatively, the chemical entity having cell killing activity may be aradioactive substance, e.g. Cobalt-60.

The effector molecule may be bound to the polypeptide of the inventionvia a covalent bond or via a non-covalent linkage. When the effectormolecule is bound to the polypeptide of the invention via a covalentbond it may be fused to the N-terminal or to the C-terminal part of thepolypeptide. Preferably, the effector molecule is fused to theC-terminal end of the second amino acid sequence to form a fusionpolypeptide. There may be one or more (e.g. two, three, four or five)amino acids between the second amino acid sequence and the amino acidsequence of the effector molecule in the fusion polypeptide.

Most preferably, the polypeptide of the invention comprises the aminoacids 2-606 of the amino acid sequence SEQ ID NO:3, or it comprises theamino acids 1-606 of the amino acid sequence SEQ ID NO:3, or it consistsof the amino acid sequence as shown in SEQ ID NO:3.

The polypeptide of the invention optionally comprises another peptide,e.g. a peptide facilitating purification, in particular a peptide beingan epitope against which an antibody is available, such as the FLAGpeptide. Purification, e.g. by means of affinity chromatography, of afusion protein comprising such a peptide is advantageous e.g. in that itmay be faster, more specific and/or gentler. The peptide may be placedat the N-terminus of the fusion protein, in between the recombinantantibody and the effector molecule, or at the C-terminus of the fusionprotein. Preferably, it is located at the N-terminus or at theC-terminus, in particular at the N-terminus. Preferably, theseconstructs also contain a cleavage site, so that the fusion protein canbe liberated therefrom, either by enzymatic cleavage, e.g. byenterokinase or by Factor Xa, or by the chemical methods known in theart. Furthermore these constructs may comprise a peptide spacerconsisting of one or more, e.g. 1 to 10, in particular about 2 aminoacids, said spacer facilitating the linkage of the above-mentionedpeptide and/or the cleavage site to the recombinant antibody. Thecleavage site is placed in such a way that the fusion protein comprisingthe recombinant antibody and the effector molecule can be easilyliberated, if desired, preferably in vitro. For example, in a proteinconstruct comprising the fusion protein designated scFv(FRP5)-ETA, theFLAG peptide and an enterokinase cleavage site are linked to a spacerand placed in front of the Fv heavy chain/light chain variable domainand exotoxin A fusion protein. If desired, the FLAG peptide can becleaved off by enterokinase, preferably after affinity purification ofthe protein, yielding a fusion protein comprising the single-chainantibody Fv(FRP5) and exotoxin A.

Another aspect of this invention is a polynucleotide encoding thepolypeptide of the invention. The term “polynucleotide(s)” generallyrefers to any polyribonucleotide or polydeoxyribonucleotide that may beunmodified RNA or DNA or modified RNA or DNA. The polynucleotide may besingle- or double-stranded DNA, single or double-stranded RNA. As usedherein, the term “polynucleotide(s)” also includes DNAs or RNAs thatcomprise one or more modified bases and/or unusual bases, such asinosine. It will be appreciated that a variety of modifications may bemade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term “polynucleotide(s)” as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including, for example, simple andcomplex cells.

Preferred are polynucleotides encoding the amino acid sequence as shownin SEQ ID NO:11. More preferably, the polynucleotide encodes apolypeptide comprising the amino acid sequence as shown in SEQ ID NO:1.Even more preferably, the polynucleotide encodes a polypeptidecomprising the amino acid sequence as shown in SEQ ID NO: 3. The mostpreferred polynucleotides comprise the nucleotide sequence as shown inSEQ ID NO:2 or the nucleotide sequence as shown in SEQ ID NO:4.

Preferably, the polynucleotide of the invention is an isolatedpolynucleotide. The term “isolated” polynucleotide refers to apolynucleotide that is substantially free from other nucleic acidsequences, such as and not limited to other chromosomal andextrachromosomal DNA and RNA. Isolated polynucleotides may be purifiedfrom a host cell. Conventional nucleic acid purification methods knownto skilled artisans may be used to obtain isolated polynucleotides. Theterm also includes recombinant polynucleotides and chemicallysynthesized polynucleotides.

Yet another aspect of the invention is a plasmid of a vector containinga polynucleotide according to the present invention. The terms “plasmid”and “vector” refer to an extrachromosomal element often carrying geneswhich are not part of the central metabolism of the cell, and usually inthe form of circular double-stranded DNA fragments. Such elements may beautonomously replicating sequences, genome integrating sequences, phageor nucleotide sequences, linear or circular, of a single- ordouble-stranded DNA or RNA, derived from any source, in which a numberof nucleotide sequences have been joined or recombined into a uniqueconstruction which is capable of introducing a promoter fragment and DNAsequence for a selected gene product along with appropriate 3′untranslated sequence into a cell. Usually, the polynucleotide in theplasmid or vector is operably linked to one or more expression controlsequences.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation. A “promoter” is a DNA sequence upstream from the start oftranscription of a gene and involved in recognition and binding of RNApolymerase and/or other proteins to initiate transcription of the gene.Usually, the promoter determines under what conditions the gene isexpressed. Usually, the promoter used herein is heterologous to thepolynucleotide of the invention to which it is operably linked.

Vectors typically perform two functions in collaboration with compatiblehost cells. One function is to facilitate the cloning of the nucleicacid that encodes the immunoglobulin variable domains, i.e. to produceusable quantities of the nucleic acid (cloning vectors). The otherfunction is to provide for replication and expression of the recombinantgene constructs in a suitable host, either by maintenance as anextrachromosomal element or by integration into the host chromosome(expression vectors). A cloning vector comprises the recombinant geneconstructs as described above, an origin of replication or anautonomously replicating sequence, dominant marker sequences and,optionally, signal sequences and additional restriction sites. Anexpression vector additionally comprises expression control sequencesessential for the transcription and translation of the recombinantgenes.

An origin of replication or an autonomously replicating sequence isprovided either by construction of the vector to include an exogeneousorigin such as derived from Simian virus 40 (SV 40) or another viralsource, or by the host cell chromosomal mechanisms.

The markers allow for selection of host cells which contain the vector.Selection markers include genes which confer resistance to heavy metalssuch as copper or to antibiotics such as geneticin (G-418), kanamycin orhygromycin, or genes which complement a genetic lesion of the host cellsuch as the absence of thymidin kinase, hypoxanthine phosphoryltransferase, dihydrofolate reductase or the like.

Signal sequences may be, for example, presequences or secretory leadersdirecting the secretion of the recombinant antibody, splice signals, orthe like. Examples for signal sequences directing the secretion of therecombinant antibody are sequences derived from the ompA gene, the pelB(pectate lyase) gene or the phoA gene.

As expression control sequences, the vector DNA comprises a promoter,sequences necessary for the initiation and termination of transcriptionand for stabilizing the mRNA and, optionally, enhancers and furtherregulatory sequences.

A wide variety of promoting sequences may be employed, depending on thenature of the host cell. Promoters that are strong and at the same timewell regulated are the most useful. Sequences for the initiation oftranslation are for example Shine-Dalgarno sequences. Sequencesnecessary for the initiation and termination of transcription and forstabilizing the mRNA are commonly available from the noncoding5′-regions and 3′-regions, respectively, of viral or eukaryotic cDNAs,e.g. from the expression host. Enhancers are transcription-stimulatingDNA sequences of viral origin, e.g. derived from Simian virus, polyomavirus, bovine papilloma virus or Moloney sarcoma virus, or of genomic,especially murine, origin.

Examples of vectors which are suitable for replication and expression inan E. coli strain are bacteriophages, for example derivatives of lambdabacteriophages, or plasmids, such as, in particular, the plasmid ColE1and its derivatives, for example pMB9, pSF2124, pBR317 or pBR322 andplasmids derived from pBR322, such as pUC9, pUCK0, pHRi148 and pLc24.Suitable vectors contain a complete replicon, a marker gene, recognitionsequences for restriction endonucleases, so that the foreign DNA and, ifappropriate, the expression control sequence can be inserted at thesesites, and optionally signal sequences and enhancers.

Microbial promoters are, for example, the strong leftward promoter PL ofbacteriophage lambda which is controlled by a temperature sensitiverepressor. Also suitable are E. coli promoters such as the lac (lactose)promoter regulated by the lac repressor and induced byisopropyl-beta-D-thiogalactoside, the trp (tryptophan) promoterregulated by the trp repressor and induced e.g. by tryptophanstarvation, and the tac (hybrid trp-lac promoter) regulated by the lacrepressor.

Vectors which are suitable for replication and expression in yeastcontain a yeast replication start and a selective genetic marker foryeast. One group of such vectors includes so-called ars sequences(autonomous replication sequences) as origin of replication. Thesevectors are retained extrachromosomally within the yeast cell after thetransformation and are replicated autonomously. Furthermore, vectorswhich contain all or part of the 2μ (2 mikron) plasmid DNA fromSaccharomyces cerevisiae can be used. Such vectors will get integratedby recombination into 2μ plasmids already existing within the cell, orreplicate autonomously. 2μ sequences are particularly suitable when hightransformation frequency and high copy numbers are to be achieved.

Expression control sequences which are suitable for expression in yeastare, for example, those of highly expressed yeast genes. Thus, thepromoters for the TRP1 gene, the ADHI or ADHII gene, acid phosphatase(PHO3 or PHO5) gene, isocytochrome gene or a promoter involved with theglycolytic pathway, such as the promoter of the enolase,glyceraldehyde-3-phosphate kinase (PGK), hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase and glucokinase genes, can be used.

Vectors suitable for replication and expression in mammalian cells arepreferably provided with promoting sequences derived from DNA of viralorigin, e.g. from Simian virus 40 (SV40), Rous sarcoma virus (RSV),adenovirus 2, bovine papilloma virus (BPV), papovavirus BK mutant (BKV),or mouse or human cytomegalovirus (CMV). Alternatively, the vectors maycomprise promoters from mammalian expression products, such as actin,collagen, myosin etc., or the native promoter and control sequenceswhich are normally associated with the desired gene sequence, i.e. theimmunoglobulin H-chain or L-chain promoter.

Preferred vectors are suitable for both procaryotic and eucaryotic hostsand are based on viral replication systems. Particularly preferred arevectors comprising Simian virus promoters, e.g. pSVgpt or pSVneo,further comprising an enhancer, e.g. an enhancer normally associatedwith the immunoglobulin gene sequences, in particular the mouse Ig H- orL-chain enhancer.

The recombinant DNA coding for a recombinant antibody of the inventioncan be prepared, for example, by culturing a transformed host cell andoptionally isolating the prepared DNA.

Another aspect of the invention are host cells that are transformed withand/or contain the plasmid or the vector according to the presentinvention. Suitable host cells include prokaryotic and eukaryotic cells(e.g. mammalian cells), yeast cells, bacterial cells (e.g. E. colicells). Preferred host cells are E. coli cells. Suitable host cells andtransformation procedures are described in U.S. Pat. No. 5,939,531 andare incorporated herein entirely by reference.

The present invention also relates to methods of culturing the hostcells and to methods for the preparation of the polypeptide of theinvention. One aspect of the invention is a process for the preparationof the polypeptide of the invention, comprising culturing a host celldescribed herein under suitable conditions and recovering thepolypeptide. Described in U.S. Pat. No. 5,939,531 are methods ofculturing host cells and methods for producing recombinant single-chainantibody fragments and antibodies. These methods are applicable to thehost cells of the present invention and to the polypeptides of thepresent invention mutatis mutandis and are incorporated herein in theirentirety by reference.

Another aspect of the invention is the use of a polypeptide according tothe invention or of a polynucleotide according to the invention for themanufacture of a medicament for the treatment of a disorder involvingaberrant activity and/or expression of ErbB2 (e.g. cancer, tumors). Suchdisorders include, but are not limited to, breast cancer, prostatecancer, ovarian cancer, squamous cell carcinoma, head and neck cancer,non small cell lung cancer, pancreas cancer, gastric cancer, salivarygland cancer, parotid tumors, melanoma, cervical carcinoma, pancreascancer, colon and colorectal cancer, bladder cancer, medulloblastoma,kidney cancer, liver cancer and stomach cancer. The disorder may also bemetastasis and/or minimal residual disease.

The invention therefore also concerns pharmaceutical compositions (e.g.for treating tumors over-expressing the growth factor receptor c-erbB-2)comprising a therapeutically effective amount of a polypeptide accordingto the invention and a pharmaceutically acceptable carrier, diluent orvehicle. Preferred are pharmaceutical compositions for parenteralapplication. Compositions for intramuscular, subcutaneous or intravenousapplication are e.g. isotonic aqueous solutions or suspensions,optionally prepared shortly before use from lyophilized or concentratedpreparations. Suspensions in oil contain as oily component thevegetable, synthetic or semi-synthetic oils customary for injectionpurposes. The pharmaceutical compositions may be sterilized and containadjuncts, e.g. for conserving, stabilizing, wetting, emulsifying orsolubilizing the ingredients, salts for the regulation of the osmoticpressure, buffer and/or compounds regulating the viscosity, e.g. sodiumcarboxycellulose, carboxymethylcellulose, sodium carboxymethylcellulose,dextran, polyvinylpyrrolidine or gelatine.

The pharmaceutical compositions of the invention contain fromapproximately 0.001% to approximately 50% of active ingredients. Theymay be in dosage unit form, such as ready-to-use ampoules or vials, oralso in lyophylized solid form.

In general, the therapeutically effective dose for mammals is betweenapproximately 0.1 and 100 μg of a polypeptide of the invention per kgbody weight, more preferably between 1 and 50 μg, and even morepreferably between 5 and 25 μg, depending on the type of polypeptide,the status of the patient and the mode of application. The specific modeof administration and the appropriate dosage will be selected by theattending physician taking into account the particulars of the patient,the state of the disease, the type of tumor treated, and the like. Thepharmaceutical compositions of the invention are prepared by methodsknown in the art, e.g. by conventional mixing, dissolving, confectioningor lyophilizing processes. Pharmaceutical compositions for injection areprocessed, filled into ampoules or vials, and sealed under asepticconditions according to methods known in the art.

In one embodiment, the polypeptide is administered in combination withanother anti-cancer therapy. Incorporated herein are all anti-cancertherapies designated as “second therapeutic agent” in the WO 03/024442A2.

In yet another aspect, the invention concerns a method for improving theproduction of a single-chain recombinant antibody directed to theextracellular domain of the receptor tyrosine kinase ErbB2, comprisingpreventing the initiation of translation from codon No. 92 of SEQ IDNO:9. This may be accomplished by modifying one or more nucleotides atpositions 262-270 of SEQ ID NO:10 such that internal translationstarting at codon No. 92 (Met 92) no longer occurs. The nucleotides maybe substituted without changing the encoded amino acid sequence.

Three non-limiting examples of the nucleotide sequence of positions262-270 are indicated in the following:

nucleotide position 262 263 264 265 266 267 268 269 270 (1) A A A A G TG A A (2) A A A T C T G A A (3) A A A T C C G A A

Alternatively, these amino acid residues Lys, Ser or Glu may be replacedby other chemically similar or unrelated amino acids, naturallyoccurring or artificially generated, and may be encoded by nucleotideswhich are chemically modified.

Another possibility is (as described hereinabove) replacing M92 in theamino acid sequence SEQ ID NO:9 with a different amino acid. Preferably,the different amino acid is serine. Yet another possibility is (asdescribed hereinabove) deleting M92 in the amino acid sequence SEQ IDNO:9 without replacing it by a different amino acid. This embodiment isrepresented by the amino acid sequence as shown in SEQ ID NO:12.

FIGURES

FIG. 1: Overview of variations of the antibody toxin scFv(FRP5)-ETA andspecific sequence characteristics

-   -   (A) Constructs for bacterial expression of scFv(FRP5)-ETA        derivatives. All expression cassettes are under the control of        the IPTG-inducible tac-promoter and code for the ErbB2-specific        scFv(FRP5), fused to exotoxin A (ETA) of Pseudomonas aeruginosa        (residues 252-613 of the wildtype toxin). Plasmid pSW220-5        carries sequences coding for an N-terminal FLAG tag (F) and two        His₆ clusters (H). In pSES212 and pSES213, the FLAG tag and the        His₆ clusters are deleted. pSES213 carries a mutation within the        scFv(FRP5) sequence changing Methionine (M) at position 92 to        Serine (S). Otherwise pSES213 is identical to pSES212. (B)        Representation of partial scFv(FRP5)-ETA DNA (SEQ ID NO:10) and        protein sequences (SEQ ID NO:9) in plasmids pSES212 and pSES213.        The change of a potential internal start-codon at codon position        92 from ATG to TCG is indicated. A sequence with moderate        similarity to a Shine-Dalgarno sequence is underlined.

FIG. 2: Expression and biological antitumoral activity of variations ofthe antibody toxin scFv(FRP5)-ETA

-   -   (A) SDS-PAGE analysis of scFv(FRP5)-ETA (220-5),        scFv(FRP5)-ETA (212) and scFv(FRP5-M92S)-ETA (213) protein        preparations. These scFv(FRP5)-ETA derivatives were expressed        in E. coli DH5α transformed with the expression plasmids        pSW220-5, pSES212 or pSES213. Inclusion bodies were isolated,        denatured and renatured. Protein samples were separated by        SDS-PAGE, and proteins were detected by Coomassie-staining.        Bands corresponding to the full-length scFv(FRP5)-ETA and        scFv(FRP5-M92S)-ETA proteins are indicated (open arrow). Major        degradation products are present in samples expressed from        plasmids pSW220-5 and pSES212 (closed arrow). In the        scFv(FRP5-M92S)-ETA (213) preparation this undesired by-product        is absent. (B) Biological activities of scFv(FRP5)-ETA (220-5)        and scFv(FRP5-M92S)-ETA (213). Murine Renca-lacZ/ErbB2 renal        carcinoma cells stably transfected with human c-erbB2 cDNA (left        panel) and ErbB2-negative Renca-lacZ control cells (right panel)        were incubated for 48 h with scFv(FRP5)-ETA (220-5) or        scFv(FRP5-M92S)-ETA (213) in triplicate samples at the indicated        concentrations. Viability of surviving cells was determined by        measuring the absorbance at 590 nm in an MTT metabolization        assay. Cells treated with PBS were used as a control. Error bars        indicate standard deviation of the mean. (C) Activity of        scFv(FRP5-M92S)-ETA (213) relative to the activity of        scFv(FRP5)-ETA (220-5). The ratios for scFv(FRP5-M92S)-ETA (213)        to scFv(FRP5)-ETA (220-5) of the A₅₉₀ values measured in (B) for        antigen-positive and antigen-negative target cells at the        indicated protein concentrations are given.

EXAMPLES Example 1 Molecular Cloning of Modified Expression Constructs

Method

Construction of scFv(FRP5)-ETA Expression Vectors

The plasmid pSW220-5 was described before (7). It contains sequencescoding for an N-terminal FLAG tag, a first His₆ cluster, theErbB2-specific scFv(FRP5), a second His₆ cluster, and truncatedPseudomonas exotoxin A (residues 252-613 of the wildtype toxin) in asingle open reading frame. Plasmid pSES211 (unpublished; provided byTopoTarget Germany AG) contains an open reading frame for scFv(FRP5)-ETAoriginally derived from pSW220-5 and still including the firstN-terminal His₆ cluster, but lacking the N-terminal FLAG tag and theinternal His₆ cluster between scFv(FRP5) and exotoxin A sequences.Plasmid pSES212 was derived by deleting the remaining N-terminal His₆cluster of pSES211. The plasmid was generated by PCR using theoligonucleotide primers 5′NdeI-scFv(FRP5)5′-CGATTAGCATATGCAGGTACAACTGCAGCAGTCAGGACC-3′ (SEQ ID NO:5) and3′XbaI-scFv(FRP5) 5′-GCTGCCGCCCTCTAGAGCTTTGATCTC-3′ (SEQ ID NO:6)(BioSpring, Frankfurt, Germany) and plasmid pSES211 as a template. Inthis reaction, the sequence of the scFv(FRP5) fragment was amplifiedwithout the coding sequences for the N-terminal His₆ cluster. The PCRproduct was subcloned into the vector pCR2.1 by TA-cloning (Invitrogen,Karlsruhe, Germany). The resulting plasmid was digested with NdeI andXhoI, and the resulting scFv(FRP5) fragment was inserted into pSES211digested with the same enzymes, yielding plasmid pSES212. The Met₉₂ toSer mutation was introduced into plasmid pSES212 by site-directedmutagenesis via PCR using the oligonucleotide primers pSES212_M92S_sense5′-CCTCAAAAGTGAAGACTCGGCTACATATTTCTGTGC-3′ (SEQ ID NO:7) andpSES212_M92S_as 5′-GCACAGAAATATGTAGCCGAGTCTTCACTTTTGAGG-3′ (SEQ ID NO:8)(BioSpring) and plasmid pSES212 as a template, yielding plasmid pSES213.The DNA sequences of all expression vectors were verified byDNA-sequencing.

Results

As a basis for the modification of the scFv(FRP5)-ETA coding region, weused the expression plasmid pSES212 (unpublished). The scFv(FRP5)-ETAcoding region within pSES212 lacks the sequences encoding the N-terminalFLAG tag and the two His₆ clusters present in the corresponding regionof pSW220-5 (see FIG. 1A). In addition, the vector backbone outside thescFv(FRP5)-ETA sequence of pSES212 is different from that of pSW220-5.These differences are not relevant in the context of this invention. Asshown in FIG. 2A (left and middle lane), the truncated scFv(FRP5)-ETAby-product is being produced to the same extent by expression usingpSES212 or pSW220-5 expression vectors. By sequence analysis, weidentified an internal ATG codon (a potential internal AUG start codonon the mRNA level) within the scFv(FRP5) antibody heavy chain framework3 region near the complementarity determining region 3 (CDR3) atnucleotide (nt) positions 274-276 of the open reading frame,corresponding to codon 92 of scFv(FRP5)-ETA in pSES212 (see FIG. 1B).This potential start codon is also preceded by a sequence with moderatesimilarity to a Shine-Dalgarno sequence at nt positions 262-270 (FIG.1B; underlined).

To test whether modification of this internal ATG/AUG codon can preventproduction of the undesired by-product, we generated the modifiedexpression plasmid pSES213. pSES213 carries a mutation changing ATG atcodon position 92 to TCG, which results in Serine (Ser; S) instead ofMethionine (Met; M) as the amino acid residue encoded by this codon (seeFIG. 1). pSES213 is otherwise identical to pSES212. Serine was chosenfor replacement of Methionine, because it is present at the equivalentposition in other murine antibody heavy chains of subgroup V (13). Inaddition, Ser and Met have similar isoelectric points, and a similarlength of their side-chains.

In another embodiment of the invention, changing codon 92 in such a waythat any amino acid other than Met is being encoded, or deleting codon92, and/or mutating the sequence from nt 262-270 in such a way that itno longer displays similarity to a Shine-Dalgarno sequence, or does nolonger act as a Shine-Dalgarno sequence, may solve the problem in asimilar fashion.

Example 2 Expression of scFv(FRP5)-ETA Derivatives in E. coli

Method

Expression of scFv(FRP5)-ETA Derivatives in E. coli, Preparation ofInclusion Bodies, Solubilization and Refolding

E. coli DH5α were transformed with the expression plasmids pSW220-5,pSES212 or pSES213. One liter expression cultures (LB, 0.5% glucose, 50μg/ml kanamycin in the case of pSES212 or pSES213, or 100 μg/mlampicillin in the case of pSW220-5) were grown at 37° C. to an OD₆₀₀ of0.8. The cultures were induced by addition of 0.5 mM IPTG for 3 hours.Cells were harvested by centrifugation (7500 g, 10 min), resuspended inPBS, and lysed in a French pressure cell. Inclusion bodies werecollected by centrifugation (10000 g, 10 min, 4° C.) and washed byresuspension in washing buffer (2 M urea, 2% Triton X-100, 500 mM NaClin PBS, pH 8) and subsequent centrifugation (10000 g, 10 min, 4° C.).Purified inclusion bodies were resuspended in solubilization buffer (8 Murea, 500 mM NaCl in PBS, pH 8). After centrifugation, the supernatantfractions were dialyzed against PBS, pH 7.4. Precipitates were removedby centrifugation (40000 g, 30 min, 4° C.) and subsequent filtrationthrough a 0.22 μm filter. Proteins were stored in aliquots at −80° C.

Results

For production of scFv(FRP5)-ETA derivatives, E. coli DH5α weretransformed with pSW220-5, pSES212 and pSES213, and selected on LB_(Kan)(for pSES212 and pSES213) or LB_(AMP) agar plates (for pSW220-5).Expression cultures of 1 l volume were inoculated with single colonies,and cultivated and induced as described in the Materials and Methodssection below. Cell pellets of these cultures were resuspended in PBS,and lysed in a French pressure cell. scFv(FRP5)-ETA proteins were mainlypresent in the insoluble fractions of these lysates (data not shown).Inclusion bodies were collected by centrifugation and washed with buffercontaining 2 M urea and 2% Triton X-100, before their denaturation withbuffer containing 8 M urea. After centrifugation, the supernatantfractions were renatured by dialysis against PBS, and the partiallypurified renatured proteins were analyzed by SDS-PAGE and subsequentCoomassie-staining (see FIG. 2A). Proteins are indicated asscFv(FRP5)-ETA or scFv(FRP5-M92S)-ETA, followed by the name of theexpression vector in brackets [scFv(FRP5)-ETA (220-5), scFv(FRP5)-ETA(212), scFv(FRP5-M92S)-ETA (213)]. For each expression culture,comparable yields of about 30-50 mg of renatured soluble protein wereobtained. As shown in FIG. 2A, the scFv(FRP5)-ETA (220-5) andscFv(FRP5)-ETA (212) protein preparations obtained contain a majorby-product (filled arrow), migrating in SDS-PAGE directly below the bandof the full-length product (open arrow). In contrast, thescFv(FRP5-M92S)-ETA (213) preparation isolated following the very sameprotocol did not contain this undesired by-product.

These data demonstrate that exchange of codon 92 from ATG to TCG, andthe corresponding amino acid residue from Methionine to Serine issurprisingly necessary and sufficient to completely prevent generationof the by-product.

Example 3 Biological Activity of scFv(FRP5-M92S)-ETA

Method

Cells and Culture Conditions

Murine renal carcinoma cells stably expressing E. coli β-galactosidase(Renca-lacZ), or β-galactosidase and human ErbB2 (Renca-lacZ/ErbB2) (8)were maintained in RPMI 1640 medium supplemented with 10%heat-inactivated FBS, 2 mM glutamine, 100 units/ml penicillin, 100 μg/mlstreptomycin, 0.25 mg/ml Zeocin (Invitrogen, Karlsruhe, Germany), and0.48 mg/ml G418 (Renca-lacZ/ErbB2).

Cell Viability Assay

Cells were seeded in 96-well plates at a density of 1.5×10⁴ cells/wellin normal growth medium. Different concentrations of scFv(FRP5)-ETAfusion proteins or diluent were added to triplicate samples, and thecells were incubated for 48 h at 37° C. in 5% CO₂ and 95% humidifiedair. An aliquot of 10 μl of 10 mg/ml MTT(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma,Deisenhofen, Germany) in PBS was added to each well, and the cells wereincubated for another 3 h. Cells were lysed by the addition of 90 μl of20% SDS in 50% dimethyl formamide, pH 4.7. After solubilization, colordevelopment due to formation of the brown formazan metabolite wasquantified by determining the absorbance at 590 nm in a microplatereader. Samples without cells served as blank.

Results

Selectivity and efficiency of tumor cell killing by scFv(FRP5)-ETAderivatives was evaluated in MTT metabolisation assays using murineRenca-lacZ/ErbB2 renal carcinoma cells expressing human ErbB2, andErbB2-negative Renca-lacZ cells as a control (8). scFv(FRP5)-ETA (220-5)protein expressed and purified in parallel with scFv(FRP5-M92S)-ETA(213) protein was used as a standard for assaying cytotoxic activities.Target cells were incubated with increasing concentrations of the fusionproteins for 48 h. After cell lysis and solubilization, cell viabilitywas quantified by determining the absorbance at 590 nm in a microplatereader (FIG. 2B). Samples without cells served as blank. At the testedconcentrations of up to 10 μg/ml, scFv(FRP5)-ETA and scFv(FRP5-M92S)-ETAfusion proteins had no effect on the survival of ErbB2-negativeRenca-lacZ cells (FIG. 2B). In contrast, scFv(FRP5)-ETA andscFv(FRP5-M92S)-ETA fusion proteins killed ErbB2-positiveRenca-lacZ/ErbB2 cells equally well and in a concentration dependentmanner (FIG. 2B). FIG. 2C represents the activity of scFv(FRP5-M92S)-ETArelative to that of scFv(FRP5)-ETA at the different concentrationstested. A value close to 1.00 indicates that there is no significantdifference. The values obtained demonstrate that scFv(FRP5-M92S)-ETA isnot significantly different in its biological activities fromscFv(FRP5)-ETA.

These data demonstrate that exchange of codon 92 from ATG to TCG, andthe corresponding amino acid residue from Methionine to Serine doessurprisingly not affect the biological activities of scFv(FRP5)-ETA. Theantitumoral activity of scFv(FRP5-M92S)-ETA is indistinguishable fromthat of scFv(FRP5)-ETA. The selectivity for ErbB2 expressing tumor cellsis also retained.

The various embodiments of this invention described herein can becombined with each other.

REFERENCES

-   1. Wels W, Harwerth I M, Mueller M, Groner B, Hynes N E. Selective    inhibition of tumor cell growth by a recombinant single-chain    antibody-toxin specific for the erbB-2 receptor. Cancer Res 1992;    52:6310-7.-   2. Spyridonidis A, Schmidt M, Bernhardt W, et al. Purging of mammary    carcinoma cells during ex vivo culture of CD34+ hematopoietic    progenitor cells with recombinant immunotoxins. Blood 1998;    91:1820-7.-   3. Schmidt M, McWatters A, White R A, et al. Synergistic interaction    between an anti-p185HER-2 Pseudomonas exotoxin fusion protein    [scFv(FRP5)-ETA] and ionizing radiation for inhibiting growth of    ovarian cancer cells that overexpress HER-2. Gynecol Oncol 2001;    80:145-55.-   4. Wels W, Beerli R, Hellmann P, et al. EGF receptor and    p185erbB-2-specific single-chain antibody toxins differ in their    cell-killing activity on tumor cells expressing both receptor    proteins. Int J Cancer 1995; 60:137-44.-   5. Azemar M, Schmidt M, Arlt F, et al. Recombinant antibody toxins    specific for ErbB2 and EGF receptor inhibit the in vitro growth of    human head and neck cancer cells and cause rapid tumor regression in    vivo. Int J Cancer 2000; 86:269-75.-   6. Wang L, Liu B, Schmidt M, Lu Y, Wels W, Fan Z. Antitumor effect    of an HER2-specific antibody-toxin fusion protein on human prostate    cancer cells. Prostate 2001; 47:21-8.-   7. Altenschmidt U, Schmidt M, Groner B, Wels W. Targeted therapy of    schwannoma cells in immunocompetent rats with an erbB2-specific    antibody-toxin. Int J Cancer 1997; 73:117-24.-   8. Maurer-Gebhard M, Schmidt M, Azemar M, et al. Systemic treatment    with a recombinant erbB-2 receptor-specific tumor toxin efficiently    reduces pulmonary metastases in mice injected with genetically    modified carcinoma cells. Cancer Res 1998; 58:2661-6.-   9. Azemar M, Djahansouzi S, Jäger E, et al. Regression of cutaneous    tumor lesions in patients intratumorally injected with a recombinant    single-chain antibody-toxin targeted to ErbB2/HER2. Breast Cancer    Res Treat 2003; 82:155-64.-   10. von Minckwitz G, Harder S, Hövelmann S, et al. Phase I clinical    study of the recombinant antibody-toxin scFv(FRP5)-ETA specific for    the ErbB2/HER2 receptor in patients with advanced solid malignomas.    Breast Cancer Res 2005; 7:R617-R26.-   11. Wels W, Harwerth I M, Zwickl M, Hardman N, Groner B, Hynes N E.    Construction, bacterial expression and characterization of a    bifunctional single-chain antibody-phosphatase fusion protein    targeted to the human erbB-2 receptor. Biotechnology (NY) 1992;    10:1128-32.-   12. Harwerth I M, Wels W, Marte B M, Hynes N E. Monoclonal    antibodies against the extracellular domain of the erbB-2 receptor    function as partial ligand agonists. J Biol Chem 1992; 267:15160-7.-   13. Kabat E A, Wu T T, Perry H M, Gottesman K S, Foeller C Sequences    of proteins of immunological interest, 5 edition, Vol. 1.    Washington: U.S. Department of Health and Human Services, 1991.

1. An isolated polypeptide comprising a first amino acid sequence whichcomprises the amino acids 2-120 of SEQ ID NO:11, and a second amino acidsequence which comprises the amino acids 136-242 of SEQ ID NO:11,wherein said first amino acid sequence and said second amino acidsequence are linked by a peptide spacer group.
 2. The polypeptideaccording to claim 1, wherein said first amino acid sequence comprisesamino acids 2-120 of SEQ ID NO:1.
 3. The polypeptide according to claim1, wherein said first amino acid sequence consists of amino acids 1-120of SEQ ID NO:1.
 4. The polypeptide according to claim 1, furthercomprising an effector molecule.
 5. The polypeptide according to claim 4wherein the effector molecule is a chemical entity having cell killingactivity.
 6. The polypeptide according to claim 5 wherein the chemicalentity having cell-killing activity is selected from the groupconsisting of chemotherapeutic drugs, cytotoxic compounds and cytostaticcompounds.
 7. The polypeptide according to claim 5 wherein the chemicalentity having cell-killing activity is a radioactive substance.
 8. Thepolypeptide according to claim 4 wherein the effector molecule is apolypeptide having cell-killing activity.
 9. The polypeptide accordingto claim 8 wherein the polypeptide having cell-killing activity is atoxin or a biologically active variant thereof.
 10. The polypeptideaccording to claim 9, wherein the toxin is Pseudomonas exotoxin or abiologically active variant thereof.
 11. The polypeptide according toclaim 1, comprising the amino acid sequence as shown in SEQ ID NO:3. 12.The isolated polypeptide of claim 1, wherein said polypeptide consistsof the amino acid sequence set forth in SEQ ID NO:3.
 13. An isolatedpolypeptide comprising the amino acid sequence set forth in SEQ IDNO:11, SEQ ID NO:1 or SEQ ID NO:12.
 14. The isolated polypeptide ofclaim 13, wherein said polypeptide consists of the amino acid sequenceset forth in SEQ ID NO:11, SEQ ID NO:1 or SEQ ID NO:12.
 15. A medicamentfor the treatment of a disorder involving aberrant activity and/orexpression of ErbB2, said medicament comprising a recombinant fusionprotein composed of a polypeptide component linked to an effectormolecule component, wherein said polypeptide component comprises theisolated polypeptide of claim 1 and the effector molecule component hascell-killing activity.
 16. The medicament according to claim 15, whereinthe disorder to be treated is a cancer.
 17. The medicament according toclaim 16, wherein the cancer is selected from the group consisting ofbreast cancer, prostate cancer, ovarian cancer, squamous cell carcinoma,head and neck cancer, non small cell lung cancer, pancreas cancer,gastric cancer, salivary gland cancer, parotid tumors, melanoma,cervical carcinoma, pancreas cancer, colon and colorectal cancer,bladder cancer, medulloblastoma, kidney cancer, liver cancer and stomachcancer.
 18. The medicament according to claim 15, wherein the effectormolecule having cell-killing activity is a polypeptide.
 19. Themedicament according to claim 18, wherein the polypeptide is a toxin ora biologically active variant thereof.
 20. The medicament according toclaim 19, wherein the toxin is Pseudomonas exotoxin or a biologicallyactive variant thereof.
 21. The medicament according to claim 15,wherein the recombinant fusion protein comprises the amino acid sequenceset forth in SEQ ID NO:3.
 22. The medicament according to claim 15,wherein the recombinant fusion protein consists of the amino acidsequence set forth in SEQ ID NO:3.